Glycoside Hydrolase Family 93

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• Author: Annabelle Varrot
• Responsible Curator: Annabelle Varrot

Glycoside Hydrolase Family GH93
Clan	GH-E
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH93.html

Substrate specificities

The characterized glycoside hydrolases of family GH93 are known to hydrolyse linear α-1,5-L-arabinan. [1, 2], EC:3.2.1-.

Kinetics and Mechanism

Figure 1. Cartoon representation of the overall structure of Arb93A (PDB ID 2W5O [2].The propeller is colored blue to red from the N- to C-terminus and arabinobiose is depicted as balls and sticks.

GH93 enzymes are exo-acting enzymes that only release arabinobiose from the non-reducing end of α-1,5-L-arabinan. These enzymes are proposed to be retaining enzymes based on the net retention of the configuration of the anomeric carbon is proposed from the products of the transglycosylation activity of the protein Abnx from Penicillium chrysogenum [3]. This proposal obtained support from the crystal structures of the Arb93A enzyme from Fusarium graminearum and Abnx both in complex with arabinobiose (Fig. 1) [2, 4]. α-L-Arabinofuranosylated pyrrolidines were shown to be good inhibitors of Arb93A. The Arb93A complex structure with a deoxyiminosugar equivalent of arabinobiose revealed a ⁴T_N twist conformation expected for the Michaelis complex, as seen for several retaining GH51 α-L-arabinofuranosidases (Fig. 2) [5]. Potent shape mimic inhibitors exploiting sp² hybridization at the anomeric carbon have been recently synthetized as well as a chromogenic substrate (Fig. 3). They are useful tools to assist further biochemical studies on L-arabinanases [6].

Catalytic Residues

From the crystal structure of Arb93A, Glu170 and Glu242 are proposed to act as catalytic nucleophile and general acid/base respectively. Mutagenesis experiment support their role in catalysis and they are strictly conserved among the family members [2]. Recent structures and mutagenesis studies for the arabinanase Abnx from Penicillium chrysogenum 31B strengthened this assignment. Mutations to alanine or glutamine of their equivalent Glu174 and Glu246 lead to inactive enzyme [4].

Three-dimensional structures

Figure 2. Electron density for deoxyiminoarabinobiose bound to the active site of Arb93A (PDB ID 2YDT [5]. Hydrogens bond are represented as dashed lines.

Figure 3. Electron density for hydroximolactone inhibitor bound to the active site of Arb93A (PDB ID 5M1Z.[6]

The crystal structure of Arb93A reveals a six-bladed β-propeller fold characteristic of GH33, GH34, and GH83 sialidases, which are also members of clan GH-E (Fig. 3) [2, 4]. The catalytic machinery is however very different from that of sialidases [7]. The wild-type structure was solved at 2.05 angstrom resolution in complex with arabinobiose . The active site is located in a deep acidic L-shaped crevice at the center of the beta-propeller (Fig. 1). Structures of the wild-type or E242A mutant enzyme in complex with iminoarabinobiose were solved at 1.6 and 1.85 angstrom resolution respectively as well as a complex with a shape mimic inhibitor and demonstrated ring distorsion (Fig. 2-3) [5, 6].

Family Firsts

First sterochemistry determination

This was determined with the Penicillium chrysogenum Abxn enzyme using ¹H-NMR to identify the transglycosylation products [3]

First catalytic nucleophile identification

This was proposed based on the structure of Fusarium graminearum Arb93A [2]

First general acid/base residue identification

This was proposed based on the structure of Fusarium graminearum Arb93A [2]

First 3-D structure

Determined for Fusarium graminearum Arb93A by Carapito and co-workers [2]

References

1. Sakamoto T and Thibault JF. (2001). Exo-arabinanase of Penicillium chrysogenum able to release arabinobiose from alpha-1,5-L-arabinan. Appl Environ Microbiol. 2001;67(7):3319-21. DOI:10.1128/AEM.67.7.3319-3321.2001 |
2. Carapito R, Imberty A, Jeltsch JM, Byrns SC, Tam PH, Lowary TL, Varrot A, and Phalip V. (2009). Molecular basis of arabinobio-hydrolase activity in phytopathogenic fungi: crystal structure and catalytic mechanism of Fusarium graminearum GH93 exo-alpha-L-arabinanase. J Biol Chem. 2009;284(18):12285-96. DOI:10.1074/jbc.M900439200 |
3. Sakamoto T, Fujita T, and Kawasaki H. (2004). Transglycosylation catalyzed by a Penicillium chrysogenum exo-1,5-alpha-L-arabinanase. Biochim Biophys Acta. 2004;1674(1):85-90. DOI:10.1016/j.bbagen.2004.06.001 |
4. Sogabe Y, Kitatani T, Yamaguchi A, Kinoshita T, Adachi H, Takano K, Inoue T, Mori Y, Matsumura H, Sakamoto T, and Tada T. (2011). High-resolution structure of exo-arabinanase from Penicillium chrysogenum. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 5):415-22. DOI:10.1107/S0907444911006299 |
5. Goddard-Borger ED, Carapito R, Jeltsch JM, Phalip V, Stick RV, and Varrot A. (2011). α-L-Arabinofuranosylated pyrrolidines as arabinanase inhibitors. Chem Commun (Camb). 2011;47(34):9684-6. DOI:10.1039/c1cc13675e |
6. Coyle T, Debowski AW, Varrot A, and Stubbs KA. (2017). Exploiting sp(2) -Hybridisation in the Development of Potent 1,5-α-l-Arabinanase Inhibitors. Chembiochem. 2017;18(11):974-978. DOI:10.1002/cbic.201700073 |
7. Gaskell A, Crennell S, and Taylor G. (1995). The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure. 1995;3(11):1197-205. DOI:10.1016/s0969-2126(01)00255-6 |